Laktoferin

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Electrophoresis. Author manuscript; available in PMC 2014 June 07.
Published in final edited form as:
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Electrophoresis. 2014 June ; 35(11): 1560–1570. doi:10.1002/elps.201300619.
Characterization of goat milk lactoferrin N-glycans and

comparison with the N-glycomes of human and bovine milk
Annabelle Le Parc1, David C. Dallas1,2, Solene Duaut1, Joelle Leonil3, Patrice Martin4, and
Daniela Barile1,2
1Department of Food Science and Technology, University of California, One Shields Avenue,
Davis, CA 95616, USA
2Foods for Health Institute, University of California, One Shields Avenue, Davis, CA 95616, USA
3INRA, UMR1253, Science and Technology of Milk and Eggs, Rennes, France
4INRA, UMR 1313, Animal Genetics and Integrative Biology, Jouy-en-Josas, France
Keywords
Goat milk; Lactoferrin; Mass spectrometry; N-linked glycan
Numerous milk components, such as lactoferrin, are recognized as health-promoting

compounds. A growing body of evidence suggests that glycans could mediate lactoferrin’s
bioactivity. Goat milk lactoferrin is a candidate for infant formula supplementation because
of its high homology with its human counterpart. The aim of this study was to characterize
the glycosylation pattern of goat milk lactoferrin. After the protein was isolated from milk
by affinity chromatography, N-glycans were enzymatically released and a complete
characterization of glycan composition was carried out by advanced mass spectrometry. The
glycosylation of goat milk lactoferrin was compared with that of human and bovine milk
glycoproteins. Nano-LC-Chip–Q-TOF MS data identified 65 structures, including high
mannose, hybrid and complex N-glycans. Among the N-glycan compositions, 37% were
sialylated and 34% were fucosylated. The results demonstrated the existence of similar
glycans in human and goat milk but also identified novel glycans in goat milk that were not
present in human milk. These data suggest that goat milk could be a source of bioactive
compounds, including lactoferrin that could be used as functional ingredients for food
products beneficial to human nutrition.
1 Introduction
Milk contains a variety of components, including proteins, endogenous peptides, lipids,
carbohydrates and minerals, all of which contribute to the growth and development of
newborns. Beyond the simple nutritional value of milk compounds, other components,
including glycoproteins, antibodies and oligosaccharides, also protect infants by reducing
Correspondence: Dr Daniela Barile, Department of Food Science and Technology, University of California, Davis, One Shields
Avenue, Davis, CA 95616, USA, dbarile@ucdavis.edu, Fax: +1-530-752-4759.
The authors have declared no conflict of interest.
Le Parc et al. Page 2
the number of pathogen infections and promoting the development of the intestinal
epithelium [1–3]. Therefore, milk is more than a simple source of essential nutrients. Milk
contains two major groups of proteins, caseins and whey proteins, both of which play a role
in the nourishment and protection of the young. During recent decades, interest has grown in
the nutritional and protective properties of whey proteins and their use as ingredients in food
products due to their high nutritional value and functional properties [4].
Lactoferrin, a member of the transferrin protein family, is one of the most abundant
glycoproteins in human and ruminant milks [5, 6]. Human milk is rich in lactoferrin, with a
concentration around 1–2 mg/mL [7–9], whereas lactoferrin concentration in ruminant milk
is 10–100 times lower than in human milk (in the range of 0.02–0.2 mg/mL) [10, 11].
Lactoferrin exhibits an array of biological activities, including antioxidant, antibacterial,
antiviral activities, iron- (and other metals) binding and immunomodulation [7, 12–14].
Lactoferrin’s function is modulated by both the polypeptide chain and its glycosylation [15].
The discovery of lactoferrin’s functional properties has resulted in increased
supplementation of bovine milk-based infant formula with bovine milk lactoferrin as a
means to enable health claims [16, 17]. Goat milk lactoferrin may better mimic the
functional properties of human milk lactoferrin and therefore be an improvement over
bovine milk lactoferrin in formula supplementation. Goat milk is an attractive source for
infant feeding due to its high digestibility, immunological properties, and its higher
concentration of minerals, including calcium and magnesium [18]. In addition, goat milk
lactoferrin has both anticancer and antimicrobial activity [19–21]. Because goat milk
oligosaccharides have many similarities to human milk oligosaccharides [22, 23], goat milk
lactoferrin glycosylation may be close to human milk lactoferrin glycosylation pattern.
Although the glycosylation profile of lactoferrin in human and bovine milk has been
described [15, 24–26], the glycosylation pattern of goat milk lactoferrin has remained
unknown.
Glycosylation is a post-translational modification with a large diversity of possible

structures. N-linked glycans (N-glycans) are glycans attached via N-acetylglucosamine
(HexNAc) to the asparagine residues of proteins in the specific amino acid sequence Asn-X-
Ser/Thr (where X can be any amino acid except proline). Literature studies report that
respectively two sites and four sites are occupied in human and bovine lactoferrin [27]. To
the best of our knowledge, there are no published reports about the actual occupancy of goat
lactoferrin’s glycosylation sites. Prediction studies reveal the presence of five potential sites
(Asn233, Asn281, Asn368, Asn476 and Asn545) just like in bovine lactoferrin [27]. The N-
glycan core is composed of two HexNAc and three mannose residues. This core is elongated
by other monosaccharides, including fucose (Fuc) and sialic acid, via the actions of
glycosyltransferases and glycosidases, which determine the degree of branching and the type
of linkage [28]. N-glycans vary widely in composition and structure, sometimes even within
a single site of glycosylation. N-glycans are divided into three main classes: high mannose,
complex and hybrid [29]. Identifying the composition, structure and glycosylation site
represents a significant analytical challenge because of the absence of template and the
presence of elongated branches and isomers. Glycosylation can modify the structural
conformation of the protein and consequently its biological activity [30, 31]. Milk glycans

can interfere with pathogen adhesion to intestinal epithelial cells [32], which strengthens the
idea that glycosylation can be involved in protecting the host against microbial and viral
attacks.
The objective of this study was to determine the profile of N-glycans of goat milk lactoferrin
by mass spectrometry. Human milk lactoferrin has a significant degree of homology (>68%)
with bovine and goat milk lactoferrin [33], so we hypothesized the presence of similar N-
glycans structures. Therefore, we used goat milk lactoferrin as a model for this investigation
because this protein shows great potential as a candidate for infant formula supplementation.
Detailed lactoferrin glycan characterization will be essential to an understanding of the
protein’s functionalities.
2 Materials and Methods

2.1 Goat milk lactoferrin purification
Goat milk was pooled from 29 goats, including five Alpines, 10 Saanens, one Toggenburg,
nine Lamanchas and four crossbreds. The lactation stage of these goats at the time of milk
collection was 104–221 days, except for one that was 462 days. Lactoferrin was purified
from goat milk by affinity chromatography as described by Barboza et al. [15], with minor
modifications. Goat milk was centrifuged at 4,000 × g for 30 min at 4°C to eliminate fat and
part of the casein micelles. After centrifugation, the aqueous phase between the upper fat
layer and the casein micelle pellet was collected. The pH of the collected fraction was
decreased to pH 4.6 by addition of hydrochloric acid to precipitate the remaining casein
micelles. As a comparison with acid precipitation, caseins were also precipitated with 200
mM CaCl2, pH 4.6, to obtain a final concentration of 60 mM CaCl2. The samples were then
centrifuged at 4000 × g for 30 min, and the supernatants were collected. The centrifugation
step was repeated and the supernatants were collected. The supernatant from acid
precipitation was concentrated 20 times using a 50 kDa molecular weight cut-off centrifugal
filter device (Amicon, Millipore, Billerica, MA, USA). The concentrate was diluted in the
affinity chromatography running buffer (100 mM Tris HCl (pH 8), or 100 mM Tris HCl (pH
8), 0.05% Tween 20, 0.05 M NaCl) and gently shaken for 30 min.
Affinity chromatography was performed by manually packing heparine Sepharose beads

(GE Healthcare Life Sciences, Pittsburgh, PA, USA) into a 12-mL polypropylene column as
a chromatographic support. The column was equilibrated with the running buffer. The
loading, washing and elution steps was performed manually on the column. The whey
protein sample was loaded onto the column. The flow-through was collected and reloaded
on the column to increase lactoferrin-binding efficiency. This step was repeated two times.
The sample was incubated with the heparin Sepharose beads for 3 h. The column was
washed with running buffer to remove non-specifically bound proteins. The bound protein
was eluted with a step-wise gradient using NaCl concentrations ranging from 0.1 to 1 M
NaCl. Fractions were collected for each salt concentration and analyzed on 12% SDS-
PAGE. Fractions with higher lactoferrin concentration (without many other protein bands)
were dialyzed (Spectra/Por® 1 dialysis tubing, MWCO 6000–8000) against water. Protein
concentrations were determined by the Bradford assay using bovine serum albumin as the
standard [34].

2.2 Protein identification

To identify the goat milk lactoferrin, the gel band with a molecular weight around 78 kDa
(the molecular weight of lactoferrin) was excised and cut into pieces. To weaken the gel,
these pieces were washed with successive baths of 100 mM NH4HCO3, pH 8, and pure
ACN for 10 min each under agitation. This step was repeated three times. The gel pieces
were incubated in a mixture of 100 mM NH4HCO3 and ACN (50:50) for 30 min. Digestion
was performed with 20 μg of trypsin (Promega, Madison, WI, USA) in 100 mM NH4HCO3,
pH 8, overnight at 37°C. Digested peptides were extracted with two baths of 5% TFA, 60%
ACN in water (v/v) for 30 min. The samples were dried overnight by vacuum centrifugation
(Genevac, Stone Ridge, NY) and resuspended in 20 μL of water prior to MS analysis.
2.3 N-glycan release and purification

Purified lactoferrin (100 μg) in water was dried and reconstituted in 100 μL of 100 mM
NH4HCO3 (pH 8), 10 mM DTT. Samples were heated for 5 min at 90°C to denature the
protein. Two microliters of 500,000 units/mL peptidyl-N-glycosidase F (New England
BioLabs, Ipswich, MA, USA) were added to the sample. The mixture was incubated
overnight at 37°C. Deglycosylated lactoferrin was removed using a C8 solid-phase
extraction column (DSC-C8 Discovery, 3-mL tube capacity, 500-mg bed weight, Supelco,
Bellefonte, PA, USA). The cartridge was conditioned with three volumes of 80% ACN,
0.1% TFA in water (v/v) and washed by three volumes of water. The sample was loaded and
the N-glycans were eluted with six volumes of water. The N-glycan solution was loaded on a
graphitized carbon cartridge (GCC-SPE, 150 mg carbon, 4-mL tube capacity, Alltech,
Deerfield, IL, USA) that was washed as described above for the C8 cartridge. The GCC
cartridge was washed with three volumes of water, and the N-glycans were eluted with three
volumes of 40% ACN, 0.1% TFA in water (v/v). The enriched N-glycan fraction was dried
overnight by vacuum centrifugation. N-glycans were rehydrated in 60 μL of water prior to
MS analysis.
2.4 Nano-LC-Chip-Q-TOF MS
The N-glycan and peptide samples were analyzed using the Agilent 6520 accurate-mass Q-
TOF LC/MS with a microfluidic nano-electrospray chip (Agilent Technologies, Santa Clara,
CA, USA). The N-glycans were separated using an HPLC-chip with a 40-nL enrichment
column and a 43-mm × 75-μm analytical column, both packed with 5 μm of porous
graphitized carbon (PGC). The peptides were separated with a C18 chip. The system was
composed of a capillary and nanoflow pump, and both used binary solvents consisting of
solvent A (3% ACN, 0.1% formic acid in water (v/v)) and solvent B (90% ACN, 0.1%
formic acid in water (v/v)). Two microliters of sample were loaded with solvent A at a
capillary pump flow rate of 4 μL/min. The N-glycan separation was performed on a 65-min
gradient delivered by the nanopump at a flow rate of 0.3 μL/min. The 65-min gradient
followed this program: 0% B (0.0–2.5 min), 0 to 16% B (2.5–20.0 min), 16 to 44% B (20.0–
30.0 min), 44 to 100% B (30.0–35.0 min) and 100% B (35.0–45.0 min). The gradient was
followed by equilibration at 0% B (45.0–65.0 min). For the peptide analysis, the gradient
was 0 to 8% B (0.0–5.0 min), 8 to 26.5% B (5.0–24.0 min), 26.5 to 100% B (24.0–48.0
min), 100% B (48.0–50.0 min) and 100% A for 10 min to re-equilibrate the column. Data

were acquired within the mass range of 450–3000 m/z for N-glycans and 500–3000 m/z for
peptides in the positive ionization mode with an acquisition rate of 2.01 spectra/s for N-
glycans and 0.63 spectra/s for peptides. An internal calibrant ion of 922.010 m/z from the
tuning mix (ESI-TOF Tuning Mix G1969–85000, Agilent Technologies) was used for
continual mass calibration. For tandem MS analysis, the N-glycans and peptides were
fragmented with nitrogen as the collision gas. Spectra were acquired within the mass range
of 100–3000 m/z. The collision energy (CE) was varied according to the equation:
in which m/z was the mass-to charge ratio of the precursor ion, k was the slope and b was
the y-intercept of the equation. The fragmentation energy was set at 1.8 V (k) with an offset
of −2.4 (b) for glycans and 3.6 V with an offset of −4.8 for peptides. Acquisition was
controlled by MassHunter Workstation Data Acquisition software (Agilent Technologies).
2.5 Peptide identification

Spectra were analyzed by a database search according to the procedure of Dallas et al. [35].
Briefly, data were exported from Agilent MassHunter in .mgf format and imported into the
offline search engine X!Tandem [36]. X!Tandem searches were against a goat milk library
compiled from previous goat milk proteome studies [37–39]. Peptides were accepted if e-
values were ≤ 0.05, corresponding to a 95% confidence level. Masses were allowed a 20
ppm error. No complete (required) modifications were included. Potential modifications
allowed were phosphorylation of serine, threonine and tyrosine; oxidation of methionine and
tryptophan; deamidation of asparagine and glutamine; and dehydration of glutamic acid. A
non-specific cleavage ([X]|[X]) (where X’ is any amino acid) was used to search against the
protein sequences. Because the instrument did not always select the monoisotopic ion for
tandem fragmentation, isotope errors were allowed (allowing up to one C13). No model
refinement was employed in X!Tandem.
2.6 Identification of the N-glycans

Compounds were identified with MassHunter Qualitative Analysis software (version B.
04.00 SP2, Agilent Technologies). The compounds were extracted using the Molecular
Feature Extractor algorithm. The software generated extracted compound chromatograms

(ECCs) in a range of 400–3,000 m/z with a ≥ 1,000 ion count cut-off, allowing charge states
of +1–3, a retention time from 5–40 min and a typical isotopic distribution of small
biological molecules. The resulting compounds were matched to a theoretical glycan library
based on previous theoretical libraries [40, 41] and previously identified milk N-glycans
from bovine and human milk [42] using a mass error tolerance of 20 ppm. The N-glycans
from the library were composed of hexose (Hex), HexNAc, Fuc, N-acetylneuraminic acid
(NeuAc) and N-glycolylneuraminic acid (NeuGc).

3 Results and discussion

Developing new products that mimic human milk properties is a current target for infant
formula manufacturers. Lactoferrin is well conserved across mammalian species; however,

the lactoferrin content is higher in human milk compared with bovine milk. This difference
stimulated an increased desire of supplementing bovine milk-based infant formula with
lactoferrin from other sources. Human lactoferrin has 68% and 70% primary sequence
homology with bovine and goat lactoferrin, respectively [33, 43]. Because goat and bovine
milk lactoferrin also have a high degree of homology (90%) [33], goat milk represents a
good candidate for infant formula supplementation. However, little information is available
on goat milk lactoferrin glycans compared with the bovine and human counterpart;
increased knowledge on goat N-glycans will enable a better understanding of the
relationship between glycan diversity and protein functionalities.
3.1 Goat milk lactoferrin purification

Obtaining a high purity lactoferrin fraction was crucial for N-glycan analysis by MS.
Achieving a high-purity sample was challenging because lactoferrin (78 kDa) is present at
relatively low levels in goat milk (concentration range of 0.02–0.2 mg/mL) and the majority
of proteins in goat milk, the caseins (a family of acidic phosphoproteins), are present at a
concentration of 26.8 g/L [44]. Due to their high concentration, caseins can easily
contaminate enriched lactoferrin fractions, and lactoferrin is known to bind to caseins [45].
Caseins are organized into a supramolecular structure, the casein micelle that is sensitive to
both acid and calcium precipitation. SDS-PAGE analysis showed that decreasing the pH to
4.6, which is the pI of the caseins, is more efficient for casein depletion than calcium
precipitation (Fig. 1A). Casein precipitation by calcium was used by Barboza et al. [15] to
eliminate casein micelles from human milk, but the lower amount of caseins in human milk
compared with goat milk [46] may explain why calcium precipitation did not successfully
remove the higher amounts of caseins in goat milk. Indeed, acid-precipitated whey sample
contained less caseins than calcium-precipitated whey sample (Fig. 1A).
After removal of milk fat by centrifugation and elimination of casein micelles by acid
precipitation, whey proteins were concentrated and smaller proteins eliminated via a 50 kDa
size-exclusion membrane filtration. This step also removed other small molecules, including
salt, lactose, oligosaccharides and endogenous milk peptides.
Lactoferrin was purified from the remaining whey proteins via heparin Sepharose affinity
chromatography. We chose this method because it is highly specific to lactoferrin [47, 48].
Indeed, despite the high similarity between lactoferrin and transferrin (also present in goat
milk) [49], only lactoferrin is retained by heparin Sepharose [48]. The purification was
optimized by modifying the physico-chemical conditions of the running buffer and elution
solutions to improve the purity of the lactoferrin fractions. The degree of purification across
these parameters was monitored by electrophoresis. Bound lactoferrin was eluted with a
step-wise gradient of increasing salt concentrations ranging from 0.1 to 1 M. The elution
profile in Fig. 1 shows a major band around 78 kDa that corresponded to the molecular
weight of the lactoferrin [50]. The majority of the lactoferrin was eluted at 0.3 M NaCl, but
various protein contaminants, including caseins, were still present (Fig. 1B). The two last

fractions (0.5 M and 1 M NaCl) contained lactoferrin with high purity; however, most of the
lactoferrin was lost in the previous fractions. To resolve this problem, the salt concentration
of the elution buffer was modified. After this optimization, most of the lactoferrin was
recovered in the 0.5 M NaCl fraction, and more contaminants were eliminated in the
previous fractions with lower concentrations of NaCl (Fig. 1C). To further improve the
purity of the lactoferrin-enriched fraction, salt (NaCl) and detergent (Tween 20) were added
to the running buffer to disrupt the potential interactions between the lactoferrin and protein
contaminants, especially residual caseins. Gel electrophoresis revealed that the addition of
salt and Tween 20 to the running buffer increased the purity of the 0.5 M and 1 M NaCl
lactoferrin fractions (Fig. 1D). This optimization showed that the combination of acid
precipitation with a single affinity chromatography step can be employed to obtain a high
purity lactoferrin fraction. Heparin Sepharose was chosen as the purification system for this
research study because it affords a highly selective binding to lactoferrin and has the ability
to deliver the level of high purity necessary to perform analytical mass spectrometry.
However, heparin Sepharose is rather expensive and other lactoferrin purification methods
are available for large-scale production. Indeed, lactoferrin has already been isolated from
bovine milk at industrial scale by ion-exchange chromatography [51].
The identity of the presumed lactoferrin band (around 78 kDa) on SDS-PAGE (Fig. 1D) was
confirmed by digesting the protein band with trypsin, analyzing the released peptides by
tandem MS (LC-MS/MS), and identifying the sequences by database searching with X!
Tandem. Twenty-six tryptic peptide sequences of goat milk lactoferrin were identified (Fig.
2A), confirming that the band represented isolated lactoferrin. An example confirmation of a
peptide identity with tandem MS is shown for the goat milk lactoferrin peptide
GSNFQLDQLQGQK in Fig. 2B.
3.2 Identification of goat milk lactoferrin N-glycans

N-glycans from the purified goat milk lactoferrin were released by enzymatic digestion,
further purified by solid-phase extraction and analyzed by nano-LC-Chip–Q-TOF MS. The
microchip, with porous graphitized carbon enrichment and analytical columns, enabled
isomer-level separation of N-glycans with reproducible retention times [52, 53].
A theoretical library for goat milk N-glycans was created based on the largest structures of
the three N-glycan types (high mannose, complex and hybrid). Theoretical libraries were
previously applied to analyze N-glycans from human and mouse serum [40, 41]. These
libraries were constructed with the three largest oligosaccharide compositions and also based
on biological rules. These structures were sequentially degraded, one monosaccharide at a
time, until the N-glycan core remained. To determine the largest putative N-glycans for goat
milk lactoferrin, knowledge of the milk N-glycome was applied [42]. The minimal level of
each monosaccharide was nine Hex, seven HexNAc, four Fuc and two sialic acids, including
NeuAc and NeuGc (Fig. 1S, Supporting Information). NeuGc was included in the library
because it was identified in glycoproteins from bovine and goat milk [42, 54, 55]. The
library contained 655 possible structures. Application of the theoretical N-glycan library as a
mass filter in Find by Molecular Feature allowed the identification of 32 different N-glycan
compositions from the goat milk lactoferrin (Table 1).

From these 32 compositions, 65 total compounds were identified, resulting from the
separation of structural and/or linkage isomers or anomers. The identified compositions
were confirmed by tandem MS as described below. Whereas isomers can be differentiated
by using tandem MS, only partial information about the possible linkage can be obtained
from mass spectrometry analysis alone. Full elucidation of glycosidic linkages requires the
combined use of pre-fractionated samples and sequential exoglycosidase enzymes with
different linkage specificity [56]. In this fashion, all N-glycans could be fully annotated.
Goat milk lactoferrin N-glycans were identified from a pool of milk from 4 different goat
breeds and, also, from crossbreds. The N-glycan pool on goat milk lactoferrin was composed
of the three main classes (high mannose, hybrid and complex) like the identified N-glycans
of human and bovine milk lactoferrin [9, 26]. Glycosylation pattern may vary among breeds.
Indeed, some studies from bovine milk revealed some variation in sialylated milk
oligosaccharides concentration between cow breeds [57]. Additionally, it has shown that
Jersey milk contains a few more neutral oligosaccharides compared to Holstein [58]. This
study revealed five high mannose glycans, nine neutral non-fucosylated hybrid/complex
glycans, six neutral fucosylated hybrid/complex glycans and 12 sialylated glycans (Table
1S, Supporting Information). The MS data showed a specific distribution of the N-glycans
across retention time. The ECCs of the N-glycans from goat milk lactoferrin are shown in
Fig. 3. High mannose glycans (green) eluted first, followed by the neutral complex/hybrid
glycans (blue). The sialylated glycans (pink) were eluted later as the concentration of ACN
in the mobile phase increased, demonstrating that their interaction with the PGC was
stronger than that of the high mannose and neutral complex/hybrid glycans. Fucosylated
compounds eluted after their non-fucosylated counterparts. These results are consistent with
previous observations of elution patterns from PGC in human and bovine N-glycans [42,
52].
Of the goat milk lactoferrin N-glycans identified, 34% were fucosylated. These results are
important because fucosylated glycans are known to be involved in pathogen inhibition [59].
In particular, Fuc-α1,2 glycans protect against Campylobacter pylori infection, a known
cause of infant diarrhea [60]. Fucosylated glycans can also exert prebiotic activity by
promoting the growth of bacteria associated with beneficial functions in the gastrointestinal
tract [61]. The presence of lactoferrin could modulate the development of a protective
intestinal microbiota because some bifidobacteria—the predominant bacteria in breast-fed
infant gut—contain enzymes that hydrolyze the N-glycan core [62]; thus, bifidobacteria
could utilize the released glycans, including fucosylated glycans. Additionally, 37% of
identified N-glycans were sialylated. Sialic acid-containing glycans protect against rotavirus
infection, which is another of main pathogens causing infant diarrhea [63].
3.3 MS/MS analysis of N-glycans

Goat milk lactoferrin N-glycans were initially identified from the mass spectra based on
accurate mass match to the library. Tandem MS was performed to confirm N-glycan
compositions. MS/MS analysis generated specific fragment ions that are common to all N-
glycans, including 163.06 m/z [Hex+H]+1, 204.09 m/z [HexNAc+H]+1 and 366.14 m/z
[HexNAc-Hex+H]+1. To facilitate data analysis, spectra were screened for the presence of
these fragment ions. The deconvoluted tandem spectra with 792.79 m/z (z = +2), which

corresponded to a hybrid-fucosylated glycan (5Hex-3HexNAc-1Fuc), is shown in Fig. 2SA,

Supporting Information. The spectrum shows the presence of the fragment ions described
above. This compound has multiple potential isomers. The isomers observed due to PGC
separation result from different enzymatic actions in the mammary epithelial cell [64]. Each
structure may differently affect lactoferrin function. Tandem mass spectra can be analyzed
for fragments that can disambiguate between various possible structures. For example, two
isomers for 792.79 m/z (z = +2) with two different positions of the Fuc residue are shown in
Fig. 2S, Supporting Information. To determine the position of Fuc, the spectra were
screened for the specific fragments represented in Fig. 2SB, Supporting Information. The
deconvoluted tandem spectrum of the glycan 5Hex-3HexNAc-1Fuc showed the presence of
the fragment 1056.37 Da, which corresponded to the mass of 3Hex- 2HexNAc-1Fuc. This
result suggests the attachment of the Fuc to the HexNAc residue in the N-glycan core rather
than to the other HexNAc of the compound, as described in Fig. 2SB, Supporting
Information.
3.4 LC-MS/MS analysis of NeuAc- and NeuGc-containing N-glycans

Data analysis revealed that goat milk lactoferrin N-glycans contained NeuAc or NeuGc.
Both NeuAc- and NeuGc-containing N-glycans are present on bovine milk proteins, but
NeuGc-containing N-glycans are not present on human milk proteins [42, 53]. In some
instances, accurate mass alone is not sufficient to positively identify N-glycan compositions.
For instance, 1Fuc-1NeuGc and 1Hex-1NeuAc have the same molecular formula and,
consequently, the same mass (453.15 Da). The composition of these glycans can be resolved
by tandem MS. The N-glycans containing sialic acids were screened for specific NeuAc or
NeuGc fragments. An MS/MS spectrum for NeuAc-containing N-glycans typically includes
fragments with 292.10 m/z [NeuAc+H]+1, 274.09 m/z [NeuAc-H2O+H]+1 and 657.23 m/z
[Hex-HexNAc-NeuAc+H]+1. A deconvoluted tandem spectrum with 865.32 m/z (z = +2),
which corresponded to the neutral mass 1728.63, is shown in Fig. 4A. This spectrum reveals
that the neutral masses of fragments corresponded to the specific monosaccharides and the
trisaccharide described previously. The composition of this compound was
5Hex-3HexNAc-1NeuAc. NeuGc-containing N-glycan tandem spectra typically include
308.10 m/z [NeuGc+H]+1, 290.10 m/z [NeuGc-H2O+H]+1 and 673.23 m/z [Hex-HexNAc-
NeuGc]+1. These fragments were observed for the compound with 1068.40 m/z (z = +2),
which corresponds to the neutral mass 2134.76 (Fig. 4B). The composition of this
compound is 4Hex-5HexNAc-1Fuc-1NeuGc.
3.5 Comparison with human and bovine milk N-glycome

The N-glycans identified in goat milk lactoferrin were compared with those in human and
bovine milk (Fig. 5). Goat milk lactoferrin resulted to have 24 and 15 N-glycans in common
with the N-glycomes of bovine and human milk, respectively, and 13 were common among
the three species. The majority of the high mannose N-glycans was conserved across these
species. Two N-glycans were identified in both human milk and goat milk lactoferrin that
were absent from bovine milk. These experiments revealed a significant degree of homology
for lactoferrin N-glycans between human and goat milk, suggesting that goat milk could be
used as a source of functional components, including lactoferrin, for infant formula
supplementation.

Differences were also observed among the three species within the hybrid and complex
structures containing Fuc and sialic acids. The number of fucosylated N-glycans of goat
milk lactoferrin was slightly lower than that on bovine milk lactoferrin (41% and 34% of all
N-glycans were fucosylated in bovine milk and goat milk lactoferrin, respectively). The
percentage of fucosylation in human milk (63/65%) [42, 53] is higher than in goat milk
lactoferrin and bovine milk. Goat milk lactoferrin contains 37.5% of sialylated N-glycans.
This value is less than the bovine milk N-glycome (43%) and close to the human milk N-
glycome (31/38%) [42, 53]. Among the 32 N-glycan compositions identified from goat milk
lactoferrin, six were absent from the bovine and human milk N-glycome. These glycans may
have unique health-improving functions that could enhance the development and protection
of the infant.
4 Concluding remarks
Current interest in lactoferrin is related to its functional roles, including antimicrobial action.
However, all the mechanisms involved in lactoferrin’s antibacterial actions have not been
well elucidated including the role of glycosylation in the protection against pathogen
infection. The N-glycans identified in this study could enable future investigations of the
effects of glycosylation on this protein’s function.
This study reveals a high diversity for N-glycan structures in goat milk lactoferrin that may
help infants’ health by decreasing pathogen infection. The analytical platform used enabled
a comprehensive profiling of N-glycans, of a protein purified from a complex mixture, with
high resolution and separation of isomeric forms. The N-glycan compositions were
identified by an automated data analysis using a theoretical library for goat milk
glycoproteins. This library was created combining biological rules and previous knowledge
on milk N-glycome; the library will find applications in the N-glycan identification for other
glycoproteins in goat milk or other sources.
As the search for novel functional foods increases, producers can turn towards other sources
of bioactive proteins than bovine milk. An abundant source of goat lactoferrin that will
enable infant formula supplementation is the significant amount of whey generated during
the production of goat cheese. This study contributed to increase the knowledge on goat
milk lactoferrin and showed that goat milk lactoferrin is a good candidate to supplement
bovine based infant formula because of the homology in the N-glycan composition with its
human counterpart. Additionally, a variety of dairy products, from cheese to yogurts, to
liquid milk could be fortified with lactoferrin to promote health in the general population.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors thank CJ Dillard for critical reading of the manuscript and Agilent Technology for assistance with the
instrumentation. The authors gratefully acknowledge Jan Carlson from the dairy goat research facility in the
Department of Animal Science, University of California, Davis, for providing the goat milk used in this study. The

authors thank INRA for partly funding this study. The authors also acknowledge support from the NIH NCCAM
(grant R01AT007079) and from the UC Davis Peter J. Shields Endowed Chair in Dairy Food Science.
Abbreviations
ECCs extracted compound chromatograms

GCC graphitized carbon column
Fuc fucose
Hex hexose
HexNAc N-acetylglucosamine
NeuAc N-acetylneuraminic acid
NeuGc N-glycolylneuraminic acid
N-glycan N-linked glycan
PGC porous graphitized carbon
References
1. Coppa GV, Zampini L, Galeazzi T, Gabrielli O. Digest Liver Dis. 2006; 38(Supplement 2):S291–
S294.
2. Yekta MA, Verdonck F, Van Den Broeck W, Goddeeris B, Cox E, Vanrompay D. Vet Med-
CZECH. 2010; 55:359–368.
3. Zivkovic AM, German JB, Lebrilla CB, Mills DA. Proc Natl Acad Sci USA. 2010; 108:4653–4658.
[PubMed: 20679197]
4. de Wit JN. J Dairy Sci. 1998; 81:597–608. [PubMed: 9565865]
5. Froehlich JW, Dodds ED, Barboza M, McJimpsey EL, Seipert RR, Francis J, An HJ, Freeman S,
German JB, Lebrilla CB. J Agric Food Chem. 2010; 58:6440–6448. [PubMed: 20415418]
6. Masson PL, Heremans JF, Dive CH. Clin Chim Acta. 1966; 14:735–739.
7. Lönnerdal B. Am J Clin Nutr. 2003; 77:1537S–1543S. [PubMed: 12812151]
8. Nagasawa T, Kiyosawa I, Kuwahara K. J Dairy Sci. 1972; 55:1651–1659. [PubMed: 4630465]
9. Smilowitz JT, Totten SM, Huang J, Grapov D, Durham HA, Lammi-Keefe CJ, Lebrilla C, German
JB. J Nutr. 2013; 12:1906–1912. [PubMed: 24047700]
10. Harmon RJ, Schanbacher FL, Ferguson LC, Smith KL. Am J Vet Res. 1975; 36:1001–1007.
[PubMed: 1096690]
11. Hiss S, Meyer T, Sauerwein H. Small Ruminant Res. 2008; 80:87–90.

12. Legrand D, Elass E, Carpentier M, Mazurier J. Cell and Mol Life Sci. 2005; 62:2549–2559.
[PubMed: 16261255]
13. Orsi N. Biometals. 2004; 17:189–196. [PubMed: 15222464]
14. Seganti L, Di Biase AM, Marchetti M, Pietrantoni A, Tinari A, Superti F. Biometals. 2004;
17:295–299. [PubMed: 15222481]
15. Barboza M, Pinzon J, Wickramasinghe S, Froehlich JW, Moeller I, Smilowitz JT, Ruhaak LR,
Huang J, Lönnerdal B, German JB. Mol Cell Proteomics. 2012; 11:015248. [PubMed: 22261723]
16. Stevenson, L.; Knowles, G. Dairy Products in Human Health and Nutrition. World Symposium;
Dairy Industry Association of Australia; p. 135-139.
17. Boland M, MacGibbon A, Hill J. Livest Prod Sci. 2001; 72:99–109.
18. Slacanac V, Bozanic R, Hardi J, Rezessyne Szabo J, Lucan M, Krstanovic V. Int J Dairy Technol.
2010; 63:171–189.
19. Atanasova J, Ivanova I. Biotechnol Biotech Eq. 2010; 24:1799–1803.

20. Kim Y, Kim MJ, Han KS, Imm JY, Oh S, Kim SH. Int J Dairy Technol. 2009; 62:277–281.
21. Recio I, Visser S. Int Dairy J. 2000; 10:597–605.
22. Martinez-Ferez A, Guadix A, Guadix E. J Membr Sci. 2006; 276:23–30.
23. Meyrand M, Dallas DC, Caillat H, Bouvier F, Martin P, Barile D. Small Ruminant Res. 2013;
113:411–420.
24. Hua S, Nwosu CC, Strum JS, Seipert RR, An HJ, Zivkovic AM, German JB, Lebrilla CB. Anal
and Bioanal Chem. 2011; 403:1291–1302. [PubMed: 21647803]
25. Nwosu CC, Strum JS, An HJ, Lebrilla CB. Anal Chem. 2010; 82:9654–9662. [PubMed:
21049935]
26. Nwosu CC, Huang J, Aldredge DL, Strum JS, Hua S, Seipert RR, Lebrilla CB. Anal Chem. 2012;
85:956–963. [PubMed: 23215446]
27. Baker EN, Baker HM. Biochimie. 2009; 91:3–10. [PubMed: 18541155]
28. Ohtsubo K, Marth JD. Cell. 2006; 126:855–867. [PubMed: 16959566]
29. Stanley, P.; Schachter, H.; Taniguchi, N. Essentials of Glycobiology. Varki, A.; Cummings, RD.;
Esko, JD.; Freeze, HH.; Stanley, P.; Bertozzi, CR.; Hart, GW.; Etzler, ME., editors. Cold Spring
Harbor Laboratory Press; Cold Spring Harbor (NY): 2009.
30. Marth JD, Grewal PK. Nat Rev Immunol. 2008; 8:874–887. [PubMed: 18846099]
31. Shental-Bechor D, Levy Y. Curr Opin Struct Biol. 2009; 19:524–533. [PubMed: 19647993]
32. Bode L. Glycobiology. 2012; 22:1147–1162. [PubMed: 22513036]
33. Leprovost F, Nocart M, Guerin G, Martin P. Biochem and Biophys Res Commun. 1994;
203:1324–1332. [PubMed: 8093048]

34. Bradford MM. Anal Biochem. 1976; 72:248–254. [PubMed: 942051]
35. Dallas DC, Guerrero A, Khaldi N, Castillo PA, Martin WF, Smilowitz JT, Bevins CL, Barile D,
German JB, Lebrilla CB. J Proteome Res. 2013; 12:2295–2304. [PubMed: 23586814]
36. Craig R, Beavis RC. Bioinformatics. 2004; 20:1466–1467. [PubMed: 14976030]
37. Cebo C, Caillat H, Bouvier F, Martin P. J Dairy Sci. 2010; 93:868–876. [PubMed: 20172206]
38. Olumee-Shabon Z, Swain T, Smith EA, Tall E, Boehmer JL. J Dairy Sci. 2013; 96:2903–2912.
[PubMed: 23498005]
39. Roncada P, Gaviraghi A, Liberatori S, Canas B, Bini L, Greppi GF. Proteomics. 2002; 2:723–726.
[PubMed: 12112854]
40. Hua S, Jeong HN, Dimapasoc LM, Kang I, Han C, Choi JS, Lebrilla CB, An HJ. Anal Chem.
2013; 85:4636–4643. [PubMed: 23534819]
41. Kronewitter SR, An HJ, de Leoz ML, Lebrilla CB, Miyamoto S, Leiserowitz GS. Proteomics.
2009; 9:2986–2994. [PubMed: 19452454]
42. Nwosu CC, Aldredge DL, Lee H, Lerno LA, Zivkovic AM, German JB, Lebrilla CB. J Proteome
Res. 2012; 11:2912–2924. [PubMed: 22439776]
43. Pakkanen R, Aalto J. Int Dairy J. 1997; 7:285–297.
44. Holt C, Jenness R. Comp Biochem Physiol Part A: Physiology. 1984; 77:275–282.
45. Anema SG, de Kruif CG. Biomacromolecules. 2011; 12:3970–3976. [PubMed: 21932853]
46. Martin, P.; Ferranti, P.; Leroux, C.; Addeo, F. Advanced Dairy Chemistry—1 Proteins. Fox, PF.;
McSweeney, PLH., editors. Springer US; 2003. p. 277-317.
47. Paramasivam M, Saravanan K, Uma K, Sharma S, Singh TP, Srinivasan A. Protein Expres Purif.
2002; 26:28–34.
48. Wang CS, Chan WY, Kloer HU. Comp Biochem Phys B. 1984; 78:575–580.
49. Park YW, Juárez M, Ramos M, Haenlein GFW. Small Ruminant Res. 2007; 68:88–113.
50. Hurley WL, Grieve RCJ, Magura CE, Hegarty HM, Zou S. J Dairy Sci. 1993; 76:377–387.
[PubMed: 8445090]
51. Tomita M, Wakabayashi H, Shin K, Yamauchi K, Yaeshima T, Iwatsuki K. Biochimie. 2009;
91:52–57. [PubMed: 18585434]
52. Chu CS, Niñonuevo MR, Clowers BH, Perkins PD, An HJ, Yin H, Killeen K, Miyamoto S, Grimm
R, Lebrilla CB. Proteomics. 2009; 9:1939–1951. [PubMed: 19288519]

53. Dallas DC, Martin WF, Strum JS, Zivkovic AM, Smilowitz JT, Underwood MA, Affolter M,
Lebrilla CB, German JB. J Agric Food Chem. 2011; 59:4255–4263. [PubMed: 21384928]
54. Addeo F, Soulier S, Pelissier JP, Chobert JM, Mercier JC, Ribadeau-Dumas B. J Dairy Res. 1978;
45:191–196. [PubMed: 670480]

55. Moreno FJ, Recio I, Olano A, Lopez-Fandino R. J Dairy Res. 2001; 68:197–208. [PubMed:
11504384]
56. Aldredge DL, Geronimo MR, Hua S, Nwosu CC, Lebrilla CB, Barile D. Glycobiology. 2013;
23:664–676. [PubMed: 23436288]
57. McJarrow P, van Amelsfort-Schoonbeek J. Int Dairy J. 2004; 14:571–579.
58. Sundekilde UK, Barile D, Meyrand M, Poulsen NA, Larsen LB, Lebrilla CB, German JB, Bertram
HC. J Agric Food Chem. 2012; 60:6188–6196. [PubMed: 22632419]
59. Newburg DS. J Animal Sci. 2009; 87:26–34.
60. Morrow AL, Ruiz-Palacios GM, Jiang X, Newburg DS. J Nut. 2005; 135:1304–1307.
61. Marcobal A, Barboza M, Froehlich JW, Block DE, German JB, Lebrilla CB, Mills DA. J Agric
Food Chem. 2010; 58:5334–5340. [PubMed: 20394371]
62. Garrido D, Dallas DC, Mills DA. Microbiology. 2013; 159:649–664. [PubMed: 23460033]
63. Yolken RH, Peterson JA, Vonderfecht SL, Fouts ET, Midthun K, Newburg DS. J Clin Invest.
1992; 90:1984–1991. [PubMed: 1331178]
64. Kunz C, Rudloff S, Baier W, Klein N, Strobel S. Annu Rev Nutr. 2000; 20:699–722. [PubMed:
10940350]

Figure 1.
Goat milk lactoferrin purification. (A) Casein removal by calcium or acid precipitation.
Caseins were precipitated from skimmed goat milk by acid precipitation at pH 4.6 or
calcium precipitation with a final calcium concentration at 60 mM. The resulting whey
fractions were analyzed by SDS-PAGE. (B–D) Optimization of elution conditions for goat
milk lactoferrin purification. The whey sample was incubated 30 min in the running buffer
and loaded on the column. The heparin Sepharose beads were washed with the running
buffer and the lactoferrin was eluted with NaCl. (B) The running buffer was 100 mM Tris
HCl, pH 8, and the lactoferrin was eluted with 0.1, 0.3, 0.5 and 1 M NaCl. (C) The running
buffer was 100 mM Tris HCl, pH 8, and the lactoferrin was eluted with 0.1, 0.2, 0.5 and 1 M
NaCl. (D) The running buffer was 100 mM Tris HCl (pH 8) 0.05% Tween 20, 0.05 M NaCl,
and lactoferrin was eluted with 0.1, 0.2, 0.5 and 1 M NaCl.

Figure 2.
Identification of goat lactoferrin by peptide mass fingerprinting. (A) Peptides identified from
lactoferrin by X!Tandem with a 95% confidence value. “Modifications” are the post-
translational modifications, including deamidation (D), oxidation (O) and dehydration (W).
(B) Deconvoluted tandem spectrum of the peptide GSNFQLDQLQGQK from goat
lactoferrin. This peptide corresponded to 731.86 m/z with z = +2.

Figure 3.
Extracted compound chromatograms (ECCs) from goat milk lactoferrin N-glycans. The
glycan types are differentiated by color: high mannose glycans are green, neutral hybrid/
complex glycans are blue, and sialylated glycans are pink.

Figure 4.
MS/MS analysis of NeuAc and NeuGc N-glycans. (A) Deconvoluted tandem spectrum of
the NeuAc-sialylated N-glycan 5Hex-3HexNAc-1NeuAc from goat milk lactoferrin. This
glycan corresponded to 865.32 m/z with z = +2. The tandem spectrum contained fragments
with neutral mass of 291.10 [NeuAc], 273.09 [NeuAc-H2O] and 656.23 [Hex-HexNAc-
NeuAc]. (B) Deconvoluted tandem spectrum of the NeuGcsialylated N-glycan
4Hex-5HexNAc-1Fuc-1NeuGc from goat milk lactoferrin. This glycan corresponded to
1068.40 m/z with z = +2. The spectrum was characterized by fragments with neutral mass of
307.09 [NeuGc], and 289.08 [NeuGc-H2O]. NeuAc, N-acetylneuraminic acid; NeuGc, N-
glycolylneuraminic acid.
Green circles, yellow circles, blue squares, red triangles, purple diamonds and gray
diamonds represent mannose, galactose, HexNAc, Fuc, NeuAc and NeuGc residues,
respectively.

Figure 5.
Comparison of goat milk lactoferrin N-glycan compositions with the known human and
bovine milk N-glycomes.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Table 1
Identified goat milk lactoferrin N-glycans.
N-glycan composition
Le Parc et al.
Mass z m/z Hex HexNAc Fuc NeuAc NeuGc Glycan type Comparison between species
1072.40 2 537.21 4 2 0 0 0 High mannose Human
1234.45 2 618.23 5 2 0 0 0 High mannose Bovine and human
1437.54 2 719.78 5 3 0 0 0 Hybrid Bovine and human
1599.59 2 800.80 6 3 0 0 0 Hybrid Bovine
1761.65 2 881.83 7 3 0 0 0 Hybrid Bovine
1802.68 2 902.35 6 4 0 0 0 Hybrid Bovine
1583.60 2 792.81 5 3 1 0 0 Hybrid-fucosylated Human
1948.74 2 975.38 6 4 1 0 0 Hybrid-fucosylated Bovine and human
1744.64 2 873.33 5 3 0 0 1 Hybrid-sialylated Goat
2052.75 2 1027.38 6 3 1 0 1 Hybrid-fucosylated- sialylated Goat
1316.51 2 659.26 3 4 0 0 0 Complex Bovine
1722.68 2 862.34 3 6 0 0 0 Complex Bovine
1462.57 2 732.29 3 4 1 0 0 Complex-fucosylated Bovine

1478.56 2 740.29 4 4 0 0 0 Complex/Hybrid Bovine and human
1681.65 2 841.83 4 5 0 0 0 Complex/Hybrid Bovine
1640.62 2 821.32 5 4 0 0 0 Complex/Hybrid Bovine and human
1624.62 2 813.32 4 4 1 0 0 Complex/Hybrid- fucosylated Bovine and human
1827.71 2 914.86 4 5 1 0 0 Complex/Hybrid- fucosylated Bovine
1786.68 2 894.35 5 4 1 0 0 Complex/Hybrid- fucosylated Bovine and human
1785.66 2 893.84 4 4 0 0 1 Complex/Hybrid- sialylated Goat
1947.72 2 974.87 5 4 0 0 1 Complex/Hybrid- sialylated Bovine
1931.73 2 966.87 5 4 0 1 0 Complex/Hybrid- sialylated Bovine and human
Page 19
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
N-glycan composition
Mass z m/z Hex HexNAc Fuc NeuAc NeuGc Glycan type Comparison between species
1728.64 2 865.33 5 3 0 1 0 Complex/Hybrid - sialylated Bovine and human
1915.73 2 958.87 4 4 1 1 0 Complex/Hybrid- fucosylated-sialylated Bovine
Le Parc et al.
2134.81 2 1068.41 4 5 1 0 1 Complex/Hybrid- fucosylated--sialylated Goat

2093.78 2 1047.90 5 4 1 0 1 Complex/Hybrid- fucosylated-sialylated Bovine
2077.78 2 1039.90 5 4 1 1 0 Complex/Hybrid- fucosylated-sialylated Bovine and human
Shown are the neutral mass, charge (z), m/z, monosaccharide composition, glycan type, and whether or not the composition was found in human and/or bovine milk. Hex, Hexose; HexNAc, N-
acetylglucosamine; Fuc, Fucose; NeuAc, N-acetylneuraminic acid, NeuGc; N-glycolylneuraminic acid.

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Electrophoresis. 2014 June ; 35(11): 1560–1570. doi:10.1002/elps.201300619.

Characterization of goat milk lactoferrin N-glycans and

Numerous milk components, such as lactoferrin, are recognized as health-promoting

Glycosylation is a post-translational modification with a large diversity of possible

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

2 Materials and Methods

Affinity chromatography was performed by manually packing heparine Sepharose beads

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

2.2 Protein identification

2.3 N-glycan release and purification

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

2.5 Peptide identification

2.6 Identification of the N-glycans

Feature Extractor algorithm. The software generated extracted compound chromatograms

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

3 Results and discussion

formula manufacturers. Lactoferrin is well conserved across mammalian species; however,

3.1 Goat milk lactoferrin purification

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

3.2 Identification of goat milk lactoferrin N-glycans

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

3.3 MS/MS analysis of N-glycans

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

corresponded to a hybrid-fucosylated glycan (5Hex-3HexNAc-1Fuc), is shown in Fig. 2SA,

3.4 LC-MS/MS analysis of NeuAc- and NeuGc-containing N-glycans

3.5 Comparison with human and bovine milk N-glycome

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

effects of glycosylation on this protein’s function.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

ECCs extracted compound chromatograms

11. Hiss S, Meyer T, Sauerwein H. Small Ruminant Res. 2008; 80:87–90.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

203:1324–1332. [PubMed: 8093048]

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

45:191–196. [PubMed: 670480]

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

Identified goat milk lactoferrin N-glycans.

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

2134.81 2 1068.41 4 5 1 0 1 Complex/Hybrid- fucosylated--sialylated Goat

Electrophoresis. Author manuscript; available in PMC 2014 June 07.

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